Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: A) Illustration of the BRCA1 polypeptide and a simplified interaction engagements. The C-terminal BRCT domain of BRCA1 interacts in a mutually exclusive manner with the phosphorylated isoforms of ABRAXAS, BACH1, or CtIP to form distinct BRCA1 complexes. In addition, BRCA1 harbors a coiled-coil motif that mediates its interaction with PALB2 and the recruitment of BRCA2 and RAD51 to sites of DNA damage. The mutant mouse alleles used in this study: Brca1tr encodes a pathogenic truncating mutation, denoted with a red arrow, that eliminates the BRCT domains. AbraxasS404A, Bach1S994A and CtipS326A each encode a missense mutation that eliminates a phosphorylation site necessary for the interaction of its protein product with the BRCT domain of BRCA1. B) Morphology and size of E13.5 embryos. The difference between wt ctrl and AABBCC was evaluated with an unpaired two-tailed student’s t-test.; wt ctrl n = 9, AABB n = 5, BBCC n = 4, AACC n = 3, AABBCC n = 3. C) Rad51 foci immunofluorescence and quantification in induced pluripotent stem (iPS) cell lines treated with 10Gy IR. Data was analyzed by one-way ANOVA.; wt ctrl n = 3, AABB n =3, BBCC n = 3, AACC n = 4, AABBCC n = 4; A = Abraxas S404A/S404A, B = Bach1 S994A/S994A, C = CtIP S326A/S326A; scale bar = 10μm. D) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cell lines, shown as a ratio of dual allele targeting in each genotype vs. control. Statistical analysis with one-way ANOVA, except for BBCC vs. AABBCC and AACC vs AABBCC, which used unpaired two-tailed student’s t-tests.; wt ctrl n = 4, AABB n = 4, BBCC n = 3, AACC n = 5, AABBCC n= 5. E) DNA fork stalling in immortalized MEFs. At least 150 DNA fibers were measured per genotype. Analysis with one-way ANOVA. F) Immunofluorescence and quantification for phospho-H2AX (S139). Foci were counted on reprogramming day 5 in > 138 cells/genotype. Statistical analysis with one-way ANOVA. Size bar = 5μm, applicable to all panels. G) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Analysis by one-way ANOVA from biological replicates. wt ctrl n = 3, AABB n = 4, BBCC n = 4, AACC n = 3, AABBCC n = 4.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER ANTIBODIES Sox2 Stemgent Cat# 09–0024 rabbit-p21 Abcam Cat# ab188224 rabbit α-alpha tubulin Abcam Cat# ab4074 Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab.

Techniques: Mutagenesis, Two Tailed Test, Immunofluorescence, CRISPR, Control, Staining

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: Related to Fig. S1. A) A schematic of Bard1-mediated SFP. The Bard1K607A point mutation prevents the recruitment of the Brca1/Bard1 heterodimer to reversed stalled replication forks, which makes them vulnerable to Mre11-dependent degradation. B) Immunofluorescence and quantification for double strand break (DSB) marker phospho - H2AX (S139). Foci were counted on reprogramming day 5 in ≥260 cells/genotype, statistical analysis with one-way ANOVA. scale bar = 5μm. C) Immunofluorescence and quantification of phospho -RPA(S33) on reprogramming day 5. Data from ≥240 cells/genotype and analyzed by one-way ANOVA.; The white arrows point to foci. scale bar = 5μm. D) Cell proliferation plots on reprogramming day 5. Arrested cells retain CFSE and are detectable as a bright peak by flow cytometry. Analysis by one-way ANOVA; wt ctrl n = 3, Brca1tr/+ n = 2, Bard1K607A/K607A n = 3. E) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Analysis with one-way ANOVA.; wt ctrl n = 6, Brca1tr/+ n = 6, Bard1K607A/+ n = 4, Bard1K607A/K607A n = 4, n=biological replicates.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER ANTIBODIES Sox2 Stemgent Cat# 09–0024 rabbit-p21 Abcam Cat# ab188224 rabbit α-alpha tubulin Abcam Cat# ab4074 Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab.

Techniques: Mutagenesis, Immunofluorescence, Marker, Flow Cytometry, Staining

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: Related to Fig. S2 A) A schematic for rescuing SFP in BRCA1 mutant cells by ablation of Smarcal1. B) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype. Analysis by one-way ANOVA. C) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype relative to control. Statistical analysis with one-way ANOVA; wt ctrl n = 9, Brca1tr/+ n = 3, Brca1tr/tr n = 3, Brca1tr/tr Smarcal1−/− n = 3. D) Immunofluorescence and quantification for double strand break (DSB) marker phospho-H2AX (S139). Foci were counted on reprogramming day 5 (≥410 cells/genotype) and statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho-RPA(S33) on reprogramming day 5. Data collected from ≥140 cells per genotype and analyzed by one-way ANOVA. F) Cell proliferation analysis with CFSE on reprogramming day 5. Statistical analysis with one-way ANOVA.; wt ctrl n = 4, Smarcal1+/− n = 4, Smarcal1−/− n = 5, Brca1tr/tr n = 2, Brca1tr/tr Smarcal1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5. Analysis by one-way ANOVA.; wt ctrl n = 3, Smarcal1+/− n = 3, Smarcal1−/− n = 3, Brca1tr/tr n = 3, Brca1tr/tr Smarcal1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Data analysis with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, Smarcal1+/− n = 4, Smarcal1−/− n = 2, Brca1tr/tr n = 12, Brca1tr/tr Smarcal1+/− n = 4, Brca1tr/tr Smarcal1−/− n = 4. n= biological replicates.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER ANTIBODIES Sox2 Stemgent Cat# 09–0024 rabbit-p21 Abcam Cat# ab188224 rabbit α-alpha tubulin Abcam Cat# ab4074 Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab.

Techniques: Mutagenesis, CRISPR, Control, Immunofluorescence, Marker, Staining

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: Related to Fig. S3. A) A schematic for rescuing HDR in Brca1 mutant cells by ablation of 53bp1. Relevant substrates that can be repaired by HDR or NHEJ are shown. B) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype to dual allele targeting in the control. Statistical analysis using one-way ANOVA, except for the comparison between wt ctrl and 53bp1−/−, evaluated with a two-tailed, unpaired student’s t-test.; wt ctrl n = 9, Brca1tr/+ n = 3, 53bp1−/− n = 5, Brca1tr/tr n = 3, Brca1tr/tr53bp1+/− n = 2, Brca1tr/tr53bp1−/− n = 4. C) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype and data was analyzed by one-way ANOVA. D) Immunofluorescence and quantification for phospho-H2AX (S139). Foci counted on reprogramming day 5 (≥410 cells/genotype), statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho RPA(S33) on reprogramming day 5. ≥140 cells per genotype, analyzed by one-way ANOVA. For control and BRCA1, images are identical for Fig. 3 and Fig. 4 for panels c and d. F) Cell proliferation analysis with CFSE dye on reprogramming day 5. Statistics with one-way ANOVA.; wt ctrl n = 4,, 53bp1’/- n = 5, 53bp1−/− n = 3, Brca1tr/tr n = 2, Brca1tr/trSmarcal1−/− n = 3, Brca1tr/tr 53bp1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5, analyzed by one-way ANOVA.; wt ctrl n = 3, 53bp1+/− n = 3, 53bp1−/− n = 2, Brca1tr/tr n = 3, Brca1tr/tr 53bp1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. Number of AP positive colonies is shown as a ratio to wild type, analyzed with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, 53bp1+/− n = 7, 53bp1−/− n = 8, Brca1tr/tr n = 12, Brca1tr/tr 53bp1−/− n = 7. I) Quantification of Nanog positive colonies in the indicated genotypes, analyzed with one-way ANOVA.; n = 3 for each genotype. J) AP staining and reprogramming efficiency quantification in human 1023 fibroblasts from adult skin biopsy in control and 53BP1 knockdown (KD) conditions. Cells were fixed on day 25 post reprogramming factor transduction, statistical analysis using an unpaired, two-tailed student’s t-test.; ctrl n = 16, 53BP1 KD n = 16. Samples with n=2 are not used for statistical comparisons. All numbers indicated are biological replicates.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER ANTIBODIES Sox2 Stemgent Cat# 09–0024 rabbit-p21 Abcam Cat# ab188224 rabbit α-alpha tubulin Abcam Cat# ab4074 Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab.

Techniques: Mutagenesis, CRISPR, Control, Comparison, Two Tailed Test, Immunofluorescence, Marker, Staining, Knockdown, Transduction

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER ANTIBODIES Sox2 Stemgent Cat# 09–0024 rabbit-p21 Abcam Cat# ab188224 rabbit α-alpha tubulin Abcam Cat# ab4074 Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab.

Techniques: Purification, Transduction, Recombinant, Knock-Out, Plasmid Preparation, Software, Imaging

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: A) Illustration of the BRCA1 polypeptide and a simplified interaction engagements. The C-terminal BRCT domain of BRCA1 interacts in a mutually exclusive manner with the phosphorylated isoforms of ABRAXAS, BACH1, or CtIP to form distinct BRCA1 complexes. In addition, BRCA1 harbors a coiled-coil motif that mediates its interaction with PALB2 and the recruitment of BRCA2 and RAD51 to sites of DNA damage. The mutant mouse alleles used in this study: Brca1tr encodes a pathogenic truncating mutation, denoted with a red arrow, that eliminates the BRCT domains. AbraxasS404A, Bach1S994A and CtipS326A each encode a missense mutation that eliminates a phosphorylation site necessary for the interaction of its protein product with the BRCT domain of BRCA1. B) Morphology and size of E13.5 embryos. The difference between wt ctrl and AABBCC was evaluated with an unpaired two-tailed student’s t-test.; wt ctrl n = 9, AABB n = 5, BBCC n = 4, AACC n = 3, AABBCC n = 3. C) Rad51 foci immunofluorescence and quantification in induced pluripotent stem (iPS) cell lines treated with 10Gy IR. Data was analyzed by one-way ANOVA.; wt ctrl n = 3, AABB n =3, BBCC n = 3, AACC n = 4, AABBCC n = 4; A = Abraxas S404A/S404A, B = Bach1 S994A/S994A, C = CtIP S326A/S326A; scale bar = 10μm. D) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cell lines, shown as a ratio of dual allele targeting in each genotype vs. control. Statistical analysis with one-way ANOVA, except for BBCC vs. AABBCC and AACC vs AABBCC, which used unpaired two-tailed student’s t-tests.; wt ctrl n = 4, AABB n = 4, BBCC n = 3, AACC n = 5, AABBCC n= 5. E) DNA fork stalling in immortalized MEFs. At least 150 DNA fibers were measured per genotype. Analysis with one-way ANOVA. F) Immunofluorescence and quantification for phospho-H2AX (S139). Foci were counted on reprogramming day 5 in > 138 cells/genotype. Statistical analysis with one-way ANOVA. Size bar = 5μm, applicable to all panels. G) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Analysis by one-way ANOVA from biological replicates. wt ctrl n = 3, AABB n = 4, BBCC n = 4, AACC n = 3, AABBCC n = 4.

Article Snippet: Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab. Does not react to S139A in human pluripotent stem cells (Orlando et al., 2021). , Millipore , Cat# 05–636.

Techniques: Mutagenesis, Two Tailed Test, Immunofluorescence, CRISPR, Control, Staining

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: Related to Fig. S1. A) A schematic of Bard1-mediated SFP. The Bard1K607A point mutation prevents the recruitment of the Brca1/Bard1 heterodimer to reversed stalled replication forks, which makes them vulnerable to Mre11-dependent degradation. B) Immunofluorescence and quantification for double strand break (DSB) marker phospho - H2AX (S139). Foci were counted on reprogramming day 5 in ≥260 cells/genotype, statistical analysis with one-way ANOVA. scale bar = 5μm. C) Immunofluorescence and quantification of phospho -RPA(S33) on reprogramming day 5. Data from ≥240 cells/genotype and analyzed by one-way ANOVA.; The white arrows point to foci. scale bar = 5μm. D) Cell proliferation plots on reprogramming day 5. Arrested cells retain CFSE and are detectable as a bright peak by flow cytometry. Analysis by one-way ANOVA; wt ctrl n = 3, Brca1tr/+ n = 2, Bard1K607A/K607A n = 3. E) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Analysis with one-way ANOVA.; wt ctrl n = 6, Brca1tr/+ n = 6, Bard1K607A/+ n = 4, Bard1K607A/K607A n = 4, n=biological replicates.

Article Snippet: Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab. Does not react to S139A in human pluripotent stem cells (Orlando et al., 2021). , Millipore , Cat# 05–636.

Techniques: Mutagenesis, Immunofluorescence, Marker, Flow Cytometry, Staining

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: Related to Fig. S2 A) A schematic for rescuing SFP in BRCA1 mutant cells by ablation of Smarcal1. B) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype. Analysis by one-way ANOVA. C) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype relative to control. Statistical analysis with one-way ANOVA; wt ctrl n = 9, Brca1tr/+ n = 3, Brca1tr/tr n = 3, Brca1tr/tr Smarcal1−/− n = 3. D) Immunofluorescence and quantification for double strand break (DSB) marker phospho-H2AX (S139). Foci were counted on reprogramming day 5 (≥410 cells/genotype) and statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho-RPA(S33) on reprogramming day 5. Data collected from ≥140 cells per genotype and analyzed by one-way ANOVA. F) Cell proliferation analysis with CFSE on reprogramming day 5. Statistical analysis with one-way ANOVA.; wt ctrl n = 4, Smarcal1+/− n = 4, Smarcal1−/− n = 5, Brca1tr/tr n = 2, Brca1tr/tr Smarcal1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5. Analysis by one-way ANOVA.; wt ctrl n = 3, Smarcal1+/− n = 3, Smarcal1−/− n = 3, Brca1tr/tr n = 3, Brca1tr/tr Smarcal1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Data analysis with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, Smarcal1+/− n = 4, Smarcal1−/− n = 2, Brca1tr/tr n = 12, Brca1tr/tr Smarcal1+/− n = 4, Brca1tr/tr Smarcal1−/− n = 4. n= biological replicates.

Article Snippet: Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab. Does not react to S139A in human pluripotent stem cells (Orlando et al., 2021). , Millipore , Cat# 05–636.

Techniques: Mutagenesis, CRISPR, Control, Immunofluorescence, Marker, Staining

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: Related to Fig. S3. A) A schematic for rescuing HDR in Brca1 mutant cells by ablation of 53bp1. Relevant substrates that can be repaired by HDR or NHEJ are shown. B) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype to dual allele targeting in the control. Statistical analysis using one-way ANOVA, except for the comparison between wt ctrl and 53bp1−/−, evaluated with a two-tailed, unpaired student’s t-test.; wt ctrl n = 9, Brca1tr/+ n = 3, 53bp1−/− n = 5, Brca1tr/tr n = 3, Brca1tr/tr53bp1+/− n = 2, Brca1tr/tr53bp1−/− n = 4. C) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype and data was analyzed by one-way ANOVA. D) Immunofluorescence and quantification for phospho-H2AX (S139). Foci counted on reprogramming day 5 (≥410 cells/genotype), statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho RPA(S33) on reprogramming day 5. ≥140 cells per genotype, analyzed by one-way ANOVA. For control and BRCA1, images are identical for Fig. 3 and Fig. 4 for panels c and d. F) Cell proliferation analysis with CFSE dye on reprogramming day 5. Statistics with one-way ANOVA.; wt ctrl n = 4,, 53bp1’/- n = 5, 53bp1−/− n = 3, Brca1tr/tr n = 2, Brca1tr/trSmarcal1−/− n = 3, Brca1tr/tr 53bp1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5, analyzed by one-way ANOVA.; wt ctrl n = 3, 53bp1+/− n = 3, 53bp1−/− n = 2, Brca1tr/tr n = 3, Brca1tr/tr 53bp1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. Number of AP positive colonies is shown as a ratio to wild type, analyzed with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, 53bp1+/− n = 7, 53bp1−/− n = 8, Brca1tr/tr n = 12, Brca1tr/tr 53bp1−/− n = 7. I) Quantification of Nanog positive colonies in the indicated genotypes, analyzed with one-way ANOVA.; n = 3 for each genotype. J) AP staining and reprogramming efficiency quantification in human 1023 fibroblasts from adult skin biopsy in control and 53BP1 knockdown (KD) conditions. Cells were fixed on day 25 post reprogramming factor transduction, statistical analysis using an unpaired, two-tailed student’s t-test.; ctrl n = 16, 53BP1 KD n = 16. Samples with n=2 are not used for statistical comparisons. All numbers indicated are biological replicates.

Article Snippet: Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab. Does not react to S139A in human pluripotent stem cells (Orlando et al., 2021). , Millipore , Cat# 05–636.

Techniques: Mutagenesis, CRISPR, Control, Comparison, Two Tailed Test, Immunofluorescence, Marker, Staining, Knockdown, Transduction

Journal: Cell reports

Article Title: BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

doi: 10.1016/j.celrep.2024.114006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-phospho-Histone H2A.X-Ser139 mouse monoclonal Ab. Does not react to S139A in human pluripotent stem cells (Orlando et al., 2021). , Millipore , Cat# 05–636.

Techniques: Purification, Transduction, Recombinant, Knock-Out, Plasmid Preparation, Software, Imaging

Journal: The EMBO Journal

Article Title: TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes

doi: 10.1038/s44318-024-00169-3

Figure Lengend Snippet: ( A ) A schematic representation of the protocol for synchronization of MCF7 cells followed by chromatin immunoprecipitation (ChIP) with indicated antibodies at the CFS and non-CFS loci. ( B , C ) Genomic organization of the FRA3B and FRA16D regions, along with the primer sets of the distal (FDR) and central (FCR) region within the FRA3B locus; FRA16D locus, is designated. ( D – F ) Endogenous Top1 but not TDP1 preferentially localizes to CFSs upon CPT (15 nM; 24 h) treatment during mitosis. Quantification of cross-linked FRA3B-FCR, FRA3B-FDR and FRA16D loci chromatin-immunoprecipitated from MCF7 cells using the specified antibodies (Top1 and TDP1). The DSB marker γH2AX antibody was used as a positive control for FRA3B and FRA16D enrichment post CPT treatment. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Data are mean ± SD, n = 3 biological replicates. ns, non-significant ( P > 0.05); ** P ≤ 0.01 (one-way ANOVA). ( G – I ) Quantification of cross-linked FRA3B-FCR, FRA3B-FDR, and FRA16D loci chromatin-immunoprecipitated from MCF7 cells transfected with FLAG-TDP1 variants (WT or S61A) using the specified antibodies (endogenous Top1). Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. ( J – L ) Quantification of cross-linked FRA3B-FCR, FRA3B-FDR, and FRA16D loci chromatin-immunoprecipitated from MCF7 cells ectopically expressing the FLAG-TDP1 variants (WT and S61A) using the specified antibodies with or without CPT treatment (15 nM; 24 h). The DSB marker γH2AX antibody was used as a positive control for FRA3B and FRA16D enrichment post CPT treatment. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Data are mean ± SD, n = 3 biological replicates. ns, non-significant ( P > 0.05); ** P ≤ 0.01 (one-way ANOVA). ( M – P ) Quantification of cross-linked β-actin, GAPDH, β2-microglobulin and β-tubulin loci chromatin-immunoprecipitated from MCF7 cells ectopically expressing the FLAG-TDP1 variants (WT and S61A) using the specified antibodies with or without CPT treatment (15 nM; 24 h). The DSB marker γH2AX antibody was used as a positive control for β-actin, GAPDH, β2-microglobulin and β-tubulin enrichment post CPT treatment. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Data are mean ± SD, n = 3 biological replicates. n.s. non-significant ( P > 0.05); * P < 0.05, ** P < 0.01; (one-way ANOVA). .

Article Snippet: Mouse monoclonal anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 , Millipore , Cat# 05-636; RRID: AB_309864.

Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Marker, Positive Control, Transfection, Expressing

Journal: The EMBO Journal

Article Title: TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes

doi: 10.1038/s44318-024-00169-3

Figure Lengend Snippet: ( A ) TDP1 −/− MEFs complemented with FLAG-TDP1 variants (TDP1 −/−/WT ; TDP1 −/−S61A ) or Empty vector (TDP1 −/−/EV ) were synchronized in mitosis and treated with or without CPT (10 μM, 1 h), released in presence of nocodazole followed by immunocytochemistry with anti-FLAG (red) to detect FLAG-TDP1 and anti-γH2AX (green) antibody. Cells were counterstained with DAPI to visualize mitotic nuclei (blue). ( B ) Quantification for the number of γH2AX foci per mitotic nucleus calculated for 50 nuclei. Data are mean ± SD, n = 3 biological replicates. * P ≤ 0.05; **** P ≤ 0.0001 (one-way ANOVA). ( C ) Representative merged image showing anaphase nucleus (blue) with γH2AX (green) foci on bridges resulting from CPT treatment in TDP1 −/−S61A MEFs. The enlarged view of the anaphase bridge has been shown. ( D ) Quantification of γH2AX-positive anaphase bridges in TDP1 −/−/EV ; TDP1 −/−/WT and TDP1 −/−S61A MEFs. Data are mean ± SD, n = 3 biological replicates. * P ≤ 0.05; ** P ≤ 0.01 (one-way ANOVA). ( E ) Representative merged image showing G1 primary nucleus (PN) with micronuclei (MN) harboring γH2AX (green) foci resulting from CPT treatment in mitosis. The enlarged view of the MN with γH2AX in TDP1 −/−S61A MEFs has been shown. ( F ) Quantification of the number of γH2AX-positive G1-MN in TDP1 −/−/EV ; TDP1 −/−/WT , and TDP1 −/−/S61A MEFs as indicated. Data are mean ± SD, n = 3 biological replicates. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 (one-way ANOVA). ( G ) TDP1 −/−/EV ; TDP1 −/−/WT , and TDP1 −/−/S61A MEFs were treated with CPT (15 nM; 9 h) in late S-phase as indicated to generate replication stress, synchronized in G2/M-phase followed by immunocytochemistry with anti-γH2AX (red) and anti-Top1cc (green) antibody. Cells were counterstained with DAPI to visualize mitotic nuclei (blue). ( H , I ) Quantifications for the number γH2AX foci ( H ) and Top1cc-positive γH2AX foci ( I ) per mitotic nucleus of TDP1 −/−/EV ; TDP1 −/−/WT and TDP1 −/−/S61A MEFs treated with CPT in late S calculated for 50 cells. Data are mean ± SD, n = 3 biological replicates. ns non-significant ( P > 0.05); * P ≤ 0.05; **** P ≤ 0.0001 (one-way ANOVA). Scale bars, 10 μm. .

Article Snippet: Mouse monoclonal anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 , Millipore , Cat# 05-636; RRID: AB_309864.

Techniques: Plasmid Preparation, Immunocytochemistry

Journal: The EMBO Journal

Article Title: TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes

doi: 10.1038/s44318-024-00169-3

Figure Lengend Snippet: ( A – D ) TDP1 −/− MEFs complemented with EV or FLAG-TDP1 variants (TDP1 −/−/WT and TDP1 −/−/S61A ) were treated with or without CPT (15 nM; 24 h) or Aph (0.4 μM, 24 h) alone or in combination (CPT + APH 24 h) and enriched at M-phase. Representative images show break-induced repair with newly synthesized mitotic DNA marked by BrdU foci (red). The γH2AX foci, signifying the DNA strand breaks, are shown in green. Cells were counterstained with DAPI to visualize nuclei (blue). ( E , F ) Quantification of γH2AX foci per nucleus and percentage of BrdU-positive mitotic nucleus obtained from immunofluorescence by confocal microscopy were calculated for 20–25 cells. Data are mean ± SD, n = 3 biological replicates. ns, non-significant ( P > 0.05); *** P ≤ 0.001; **** P ≤ 0.0001 (one-way ANOVA). ( G ) Chromatin fractions were prepared from TDP1 −/−/WT and TDP1 −/−/S61A MEFs after CPT treatment (15 nM; 24 h) and analyzed by western blotting to detect FLAG-TDP1 variants and MUS81 with anti-FLAG and anti-MUS81 antibodies, respectively. Anti-HH3 and anti-pHH3 were used as chromosomal and mitotic markers. Protein levels of MUS81, FLAG-TDP1 WT , and FLAG-TDP1 S61A were analyzed in whole-cell lysates (WCE) to ensure equal levels of protein before chromatin fractionation. ( H ) Quantification showing the relative chromosomal enrichment of FLAG-TDP1 variants (WT or S61A) in interphases (Asn) and mitotic (M) chromosomes (left panel). Quantification showing the relative enrichment of MUS81 on mitotic chromosomes in interphases (Asn) and mitosis (M) for indicated cells treated with CPT (15 nM; 24 h) (right panel) n = 3 biological replicates. Data are mean ± SD, n = 3 biological replicates. ns, non-significant ( P > 0.05); * P ≤ 0.05; ** P ≤ 0.01 (one-way ANOVA). ( I ) Representative images of immunofluorescence microscopy showing induction of CPT (15 nM, 24 h)-induced γH2AX (green) and MUS81 (red) foci during mitosis in TDP1 −/−/EV , TDP1 −/−/WT , and TDP1 −/−/S61A MEFs, co-transfected either with Si Ctrl or Si MUS81 to knockdown MUS81. Cells were counterstained with DAPI to visualize mitotic nuclei (blue). Note: Colocalization of CPT-induced γH2AX and MUS81 foci in merged images indicates MUS81 overloading amplifies mitotic DNA breaks in TDP1 −/−/S61A MEFs. ( J ) Representative western blot analysis for the immunofluorescence microscopy in ( I ) to detect the levels of MUS81 and FLAG-TDP1 in TDP1 −/−/EV , TDP1 −/−/WT , and TDP1 −/−/S61A MEFs, co-transfected either with Si Ctrl or Si MUS81 to knockdown MUS81. β-actin has been used as loading control. ( K ) Quantifications of MUS81 foci on the mitotic chromosomes scored for 50 nuclei (each category) as depicted by the corresponding bar diagram. Data are mean ± SD, n = 3 biological replicates. ns, non-significant ( P > 0.05); ** P ≤ 0.01 (one-way ANOVA). ( L ) Quantifications for CPT-induced γH2AX foci on mitotic nuclei in indicated cells ( n = 50 cells from three biological replicates; mean ± SD). Note: siRNA knockdown of MUS81 significantly reduced CPT-induced γH2AX. ns, non-significant ( P > 0.05); ****P ≤ 0.0001 (one-way ANOVA). ( M ) A schematic representation for the protocol followed for the ChIP of endogenous MUS81 at the CFS loci in mitosis following CPT (15 nM, 24 h) treatment. ( N – P ) Quantification of cross-linked FRA3B-FCR, FRA3B-FDR, and FRA16D loci chromatin-immunoprecipitated from MCF7 cells transfected with empty vector (EV) or FLAG-TDP1 variants (WT or S61A) using the specified antibodies. Fold enrichment over IgG was determined and is shown for each primer pair for the ChIP. Data are mean ± SD, n = 3 biological replicates. * P ≤ 0.1; ** P ≤ 0.01 (one-way ANOVA). Scale bars, 10 μm. .

Article Snippet: Mouse monoclonal anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 , Millipore , Cat# 05-636; RRID: AB_309864.

Techniques: Synthesized, Immunofluorescence, Confocal Microscopy, Western Blot, Fractionation, Microscopy, Transfection, Knockdown, Control, Immunoprecipitation, Plasmid Preparation

Journal: The EMBO Journal

Article Title: TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes

doi: 10.1038/s44318-024-00169-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse monoclonal anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 , Millipore , Cat# 05-636; RRID: AB_309864.

Techniques: Virus, Recombinant, Transfection, Modification, Protease Inhibitor, Reverse Transcription, Mutagenesis, Imaging, Software

Journal: bioRxiv

Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

doi: 10.1101/2024.06.25.600517

Figure Lengend Snippet: a) Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b) Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c) Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. d) Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL FMLV VLP per 100 µL media. Data are averaged across 3-6 replicates per cell type, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.

Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

Techniques: Derivative Assay, Transduction, Labeling, Amplification

Journal: bioRxiv

Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

doi: 10.1101/2024.06.25.600517

Figure Lengend Snippet: a) Unmerged panels from , showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons. For a-b: Neurons transduced 2 weeks into differentiation, and imaged 3 days post-transduction. DSBs are co-labeled by markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Scale bar is 20 µm. b) Additional representative ICC images showing DSBs induced by Cas9-VLPs in human iPSC-derived neurons, compared to age-matched untransduced neurons.

Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

Techniques: Derivative Assay, Transduction, Labeling

Journal: bioRxiv

Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

doi: 10.1101/2024.06.25.600517

Figure Lengend Snippet: a-c) DSB repair markers over time in untransduced (a), B2Mg1-transduced (b), and NEFLg1-transduced (c) neurons. DSBs are co-labeled by ICC markers γH2AX (red) and 53BP1 (green). Dose: 1 µL FMLV VLP per 100 µL media. Neurons were fixed at 1,4, or 7 days post-transduction as labeled. One representative image from each condition is shown. Transduction was 2 weeks into differentiation. Scale bar is 20 µm. Same experiment as , but now showing unmerged panels individually, and including additional conditions (timepoints, sgRNAs). Therefore, the merged panels for untransduced and B2Mg1-transduced at 1 day and 7 days are the same as in , but uncropped here.

Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

Techniques: Labeling, Transduction

Journal: bioRxiv

Article Title: Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

doi: 10.1101/2024.06.25.600517

Figure Lengend Snippet: a) ChIP-qPCR for γH2AX binding at various distances from the cut site over time. Same conditions as , but with γH2AX antibody instead of Mre11. b) Schematic illustrating our strategy to detect cut-and-resealed loci by using a ChIP-qPCR amplicon that spans across the cut site. Repair protein binding suggests that the locus had been cut, and successful PCR amplification suggests that the cut has since been resealed. Note: however, it remains ambiguous whether these loci were sealed with or without an indel. c-d) Some loci have been resealed as early as 2 days post-transduction. ChIP-qPCR using the spanning amplicon to detect cut-and-resealed loci, with both Mre11 (c) and gH2AX (d). Same procedures as and S9a, but using different amplicons (cut site spanning, and different chromosome control). e) Cas9 protein in iPSCs gets quickly diluted and/or degraded to background levels within 2 days post-transduction; therefore, these neuron ChIP-qPCR data cannot be compared to iPSCs. Pulse-chase to track degradation of Halo-tagged Cas9 in iPSCs. First, iPSCs (with/without lentivirally integrated Halo-Cas9 and B2Mg1) were seeded onto glass-bottom 96-well plates with ∼2,000 cells per well. iPSCs were pulsed with 40 µM fluorescent Halo ligand (Promega HaloTag-JF549, cat. #GA1110) for 1 hour, then washed with fresh media 3 times to prevent newly translated Cas9 protein from being labeled. iPSCs were then chased with 2 µM of an unlabeled Halo ligand (Promega ent-HaloPROTAC3, cat. #GA4110) as a binding competitor. Nuclei were labeled with NucBlue (ThermoFisher, cat. #R37605) 20 min before live cell imaging on the Image Xpress Confocal Microscope. Halo fluorescence signal was measured at several timepoints to track the degradation/dilution of the pulse-labeled Cas9 molecules over time. Analyzed in CellProfiler. 8 replicate wells; error bars show standard deviation.

Article Snippet: Then, neurons were incubated with the following buffers at RT, with two PBS washes after each incubation: primary antibody solution for 1 hour (Mouse Anti-phospho-Histone H2A.X Ser139 Antibody, clone JBW301, Sigma #05-636, 1:4000 diluted in blocking buffer; Rabbit Anti-53BP1 Antibody, Novus #100-305, 1:1000 diluted), secondary antibody solution for 1 hour (Goat anti-Mouse IgG H+L 568, Invitrogen #A-11031; Goat anti-Rabbit IgG H+L 488, Invitrogen #A-11034; both 1:1000 diluted in blocking buffer), then DAPI for 2 minutes (1:1000 diluted in PBS, Thermo #62248).

Techniques: Binding Assay, Amplification, Protein Binding, Transduction, Control, Pulse Chase, Labeling, Live Cell Imaging, Microscopy, Fluorescence, Standard Deviation